Human Papillomavirus (HPV) is the most common sexually transmitted infection. In Italy, HPV vaccination is now offered free of charge to 12-year-old females. However, some regional health authorities have extended free vaccination to other age-groups, especially to girls under 18 years of age. We conducted a multicentre epidemiological study to ascertain the prevalence of different genotypes of HPV in young Italian women with normal cytology, with the aim of evaluating the possibility of extending vaccination to older females.The study was performed in 2010. Women aged 16–26 years with normal cytology were studied. Cervical samples were analyzed to identify the presence of HPV by PCR amplification of a segment of ORF L1 (450 bp). All positive HPV-DNA samples underwent viral genotype analysis by means of a restriction fragment length polymorphism assay.Positivity for at least one HPV genotype was found in 18.2% of the 566 women recruited: 48.1% in the 16–17 age-class, 15.4 in the 18–20 age-class, 21.9% in the 21–23 age-class, and 15.5% in the 24–26 age-class; 10.1% of women were infected by at least one high-risk HPV genotype. HPV-16 was the most prevalent genotype. Only 4 (0.7%), 4 (0.7%) and 3 (0.5%) women were infected by HPV-18, HPV-6 and HPV-11, respectively. Of the HPV-DNA-positive women, 64.1% presented only one viral genotype, while 24.3% had multiple infections. The HPV genotypes most often involved in multiple infections were high-risk. A high prevalence was noted in the first years of sexual activity (48.1% of HPV-DNA-positive women aged 16–17 years); HPV prevalence subsequently declined and stabilized.The estimate of cumulative proportions of young women free from any HPV infection at each age was evaluated; 93.3% and 97.1% of 26 year-old women proved free from HPV-16 and/or HPV-18 and from HPV-6 and/or HPV-11, respectively.Our findings confirm the crucial importance of conducting studies on women without cytological damage, in order to optimise and up-date preventive interventions against HPV infection, and suggest that vaccinating 26-year-old females at the time of their first pap-test is to be recommend, though this issue should be further explored. Many types of human papillomavirus (HPV) are known. Of these, about 50 have a high tropism for the ano-genital mucosa and are sexually transmitted. According to the most recent classification of the International Agency for Research on Cancer (IARC) 12 genotypes are defined as high-risk (HR) oncogenic . Moreover, a probable oncogenic risk has been assigned to genotype 68, and a possible oncogenic risk to the types: HPV-26, 53, 66, 67, 70, 73, 82, 30, 34, 69, 85, and 97. Many low-risk (LR) genotypes that cause benign lesions and warts are also known  [After zur Hausen’s discovery of the association between persistent infection by oncogenic types of HPV and cervical cancer , epidemiThe prevalence of HPV in subjects with lesions displays a different distribution by region and age -9. The fSo far, two HPV vaccines have been licensed: a bivalent vaccine  and a quadrivalent vaccine (Gardasil® Merck and Co). Both have a prophylactic indication and are able to prevent pre-cancerous lesions and cancers due to persistent infection by HPV-16 and HPV-18. Furthermore, both vaccines have been shown to elicit cross-protection against other high-risk HPV types ,26. GardThe epidemiological data collected worldwide constitute the starting point for vaccination strategies. However, in order to draw up optimal vaccination policies, it is crucial to know the prevalence of the different HPV types in the overall population, and not only in subjects with lesions or cancer. Indeed, such knowledge is essential to predicting the expected impact and monitoring the actual impact of HPV immunisation. For this reason, several extensive studies on women with normal cytology have been conducted ,27-29.The World Health Organization and the European Centre for Disease Prevention and Control recommend HPV vaccination and indicate young females (aged 10–14 years) as the target population ,31. In ETwo years after the start of HPV vaccination, we conducted a multicentre epidemiological study to ascertain the prevalence of different genotypes of HPV in young Italian women with normal cytology, with a view to evaluating the possibility of extending vaccination – for instance by offering free vaccination to 26-year-old females.The study protocol, which was used by all research units, was approved by the Ethics Committee of the Local Healthcare Unit (LHU) in Genoa, Italy.The study was performed in 2010 and enrolment was carried out during the first six months of the year.The study population was recruited in three cities in northern Italy .st January to 30th June 2010. We chose this age-group because it is potentially the best target for vaccination. Written informed consent was obtained from every participant. Women were considered eligible for enrolment if they were sexually active, were not pregnant, had not been vaccinated against HPV and had no previous history of cervical abnormalities.The study protocol involved enrolling all consecutive eligible young women aged 16–26 years who spontaneously accessed gynaecology centres of the LHUs for medical consultations from 1All participants underwent a Pap smear and HPV-DNA test. Subsequently, only women with normal cytologically negative Pap smears were studied, as the aim of the study was to evaluate the prevalence of HPV genotypes in healthy young women without any HPV lesions.Women with a positive pap smear were excluded from the study. They were followed up by the gynaecology centres of the LHUs or by their own gynaecologists and were treated in accordance with the Italian guidelines . No dataCervical samples were collected by means of a spatula, immersed and rinsed in a vial containing 20 ml of PreservCyt® solution  and stored at room temperature (RT) until processing. All samples were analysed in the molecular laboratory of the Department of Biomedical Sciences for Health – University of Milan.Ten millilitres of each PreservCyt® solution containing cervical cells was centrifuged at 3800 g for 10 min at RT. Cellular pellets were resuspended in 1 ml Phosphate Buffered Saline (PBS) and stored at −20°C until molecular analyses were carried out.® EasyMAG®, bioMérieux, Lyon, France) according to the manufacturer’s instructions. The concentration and purity of the DNA extracted were evaluated by means of a spectrophotometer . DNA integrity was assessed by amplification of a 268 base pair (bp) fragment in the ubiquitous β-globin gene by using the primer pair PCO4 and GH20[DNA was extracted from cervical samples by means of a commercial method  restriction enzymes diluted in their respective buffers for 1 h at 37°C. The digestion products were identified following separation in 3% agarose gels, and restriction patterns were compared with appropriate standards . The pattern of fragments generated by the three restriction enzymes enabled the genotype to be identified [Amplified products (1 g each) were added to three different digestion solutions, each containing 1U of either entified ,39.Samples displaying complex or undetermined RFLP patterns were retested. Samples that could not be assigned with confidence to a certain type after two consecutive analyses were classified as “not typing” (NT).The prevalence of HPV-infected cases, together with their 95% Confidence Intervals (CI), was described by age-class and city of residence, and the Chi-square test was used to analyze the differences . The subjects enrolled were broken down into 4 age-classes: 16–17, 18–20, 21–23 and 24–26 years old. In addition, the percentage of infections, broken down by risk-group in the different age-classes, was calculated on considering the total number of infections detected in each age-class.The Kaplan-Meier method was used to estimate the cumulative proportions of young females who were free from HPV infection at each age, and graphic descriptions of all HPV genotypes and different HPV genotypes were drawn up. All analyses were performed with the Statistical Package for Social Sciences, for Windows, version 16.0 .A total of 650 women who met the inclusion criteria were invited to participate in the study; 16 declined. As few women refused to participate, the reasons for refusal were not investigated.Of the 634 young women who agreed to take part in the study, 566 with normal cervical cytology were recruited. The 68 who were excluded had a positive Pap smear. In Milan, Genoa and Turin 281, 156 and 129 subjects were enrolled, respectively. Table The mean age of the participants on enrolment was 19.7 years (SD = 2.4). Positivity for at least one HPV genotype was found in 103 women . In Milan, Genoa and Turin, 16.0% (95% CI 11.3-20.8), 24.4% (95% CI 17.8-31.9) and 15.5% (95% CI 9.3-22.9) of women, respectively, were HPV-positive. No statistically significant differences emerged among the cities of residence (p = 0.064). Among the 103 HPV-DNA-positive women, the samples from 12 subjects (11.6%) were NT.Regarding high-risk HPV types, fifty-seven women (55.3%) were infected by at least one high-risk HPV genotype. HPV-16 was the most prevalent genotype, being detected in 16 women (15.5%). Only 4 subjects (3.9%) were infected by HPV-18. Sixty-six subjects (64.1%) presented only one viral genotype, while 25 (24.3%) had multiple infections. Globally, 132 HPV infections were detected in the 103 HPV DNA-positive women. Figure Regarding the importance of the different genotypes on considering the infection type (single or multiple) we found that most of the single infections were caused by high-risk or probable/possible high-risk HPV genotypes (74.2%). Concomitant HPV-16 and HPV-18 infections were not found.Figure Regarding low-risk HPV types, HPV-40 was the genotype most often detected. Only 4 (3.9%) and 3 (2.9%) women were infected by HPV-6 and HPV-11, respectively. Of 25 multiple infections, only one was caused exclusively by low-risk HPV genotypes (HPV-6 and HPV-11 together).Table Figure Figure Figure Our study revealed that 18.2% of the sexually active females with normal cervical cytology were infected by at least one HPV genotype; this result is consistent with data obtained by other authors in women without lesions ,28. In 2A prevalence study on HPV infection in women aged 18–24 years was carried out in 2006–2007 in Italy. The authors found a prevalence of HPV of about 30% and, on considering only the high-risk genotypes, a prevalence of approximately 20%. These values are higher than ours. However, their study population included women with normal cytology and women with cytological lesions. On considering only the data from the women with negative cytology, the authors found a prevalence of about 20%, which is comparable to our findings .We recorded a prevalence of high-risk HPV genotypes of 10.1%, while Clifford et al. found a prevalence of high-risk HPV genotypes of 5.2% in Europe. However, these authors considered a very large age-range, namely 15–74 years .Regarding the relative importance of different high-risk HPV types, the 6 most important in our study were: HPV-16, -52, -56, -31, -51, -18. Our results are similar, but not identical, to those reported by other authors. In Europe in 2005, Clifford et al. reported the following high-risk HPV types: HPV-16, -31, -18, -56, -45 and −35 , and de Another study that provided robust baseline estimates of the prevalence and distribution of HPV types among a nationally representative sample of women (25–64 years old) in England, prior to any impact of the National HPV Immunisation Programme, indicated that the most frequent HPV genotypes among subjects with normal cytology were: HPV-16, -52, -31 and -45 . These dAmong the HPV genotypes recently defined as probable/possible high-risk, we most frequently identified type 66, while among low-risk viruses type 40 was most frequently identified.Regarding multiple infections, which are associated with a higher risk of developing high-degree precancerous lesions, we found a higher prevalence (24.3%) than other authors ,45. For In young women (20–29 years old) randomly selected from a population of Danish women, Nielsen et al. found a prevalence of multiple infections of 10.5% . The difFurthermore, in our study, although the subjects recruited were not distributed homogeneously by age, the highest percentage of multiple infections  was seen among 16-17-year-old subjects. This result is consistent with the data from the literature, and could be explained by the sexual habits of younger women and by the fact that they have less immune experience than older women ,49.We found statistically significant differences in the HPV prevalence rates observed at different ages, a particularly high prevalence being noted in the first years of sexual activity. In the subsequent age-classes, the prevalence declined and stabilized. These results are supported by substantial data that indicate that the first HPV infections often occur soon after the first sexual intercourse -53. In oRegarding the estimated cumulative proportions of young females without cervical lesions who were free from any HPV infection at each age, it is interesting that 49.8% of subjects were free from any HPV infection at the age of 26 years Figure A. Since In Italy, HPV vaccination has been offered free of charge to 12-year-old females since 2008, and both the quadrivalent and the bivalent vaccine are available. More recently, some Italian Regions have extended free vaccination to other female cohorts. Although the Italian Ministry of Health initially recommended vaccination for women aged 25–26 years, too , most ItIn this perspective, our study suggests that vaccinating females without cervical lesions up to 26 years of age appears to be useful both from the point of view of public health and from the point of view of the individual health of the women. Furthermore, studies on HPV vaccines have demonstrated that vaccination is effective, albeit to different degrees, both in naïve females and in women with evidence of prior HPV exposure .Our considerations regarding the need to extend free vaccination to women up to 26 years of age are supported by epidemiological and economic studies. Epidemiological studies have shown that the risk of HPV infection remains in older age-groups; indeed, all sexually active women are exposed to the risk of infection at any age ,65. A meOur study confirms the importance of type 16 in both single and multiple infections and highlights the importance of type 52, which was second only to type 16 in women without cytological damage. Furthermore, it confirms the crucial importance of conducting studies on women without cytological damage, in order to optimise and up-date preventive interventions against HPV infection, such as vaccination.Our findings also emphasize the importance of vaccinating young women up to 18 years of age through catch-up campaigns. Furthermore, they suggest the utility of updating vaccination strategies so as to include women up to 26 years of age, while taking the opportunity to perform a Pap test at the time of administering the first dose of vaccine. Nevertheless, this strategy should be further explored. This strategy could help to achieve good coverage, raise awareness of cervical cancer prevention and monitor the effectiveness of the vaccine at subsequent screening visits.HPV: Human papillomavirus; IARC: International Agency for Research on Cancer; HR: High-risk; LR: Low-risk; LHU: Local Healthcare Unit; PBS: Phosphate buffered saline; NT: Not typing; CI: Confidence interval.The authors declare that they have no competing interests.RG coordinated and supervised the research. RG, DP, ET and CMZ designed the study. RG and DP coordinated the Genoa unit’s research. ET coordinated the Milan unit’s research and the laboratory activities. CMZ coordinated the Turin unit’s research. SB and ERF carried out the laboratory analyses and performed the first data-quality control. PLL, AB and DA optimized the informatics database and performed the final data-quality control. RG and DP performed the first statistical analyses. SR supervised the first statistical analyses and conducted the final statistical analyses. RG, DP, DA and SR evaluated the results. RG, DP and DA wrote the manuscript. All authors revised the manuscript and contributed to improving the paper. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/13/575/prepub


Pseudomonas sp. SH-C52 from the rhizosphere of sugar beet plants grown in a soil suppressive to the fungal pathogen Rhizoctonia solani and showed that its antifungal activity is, in part, attributed to the production of the chlorinated 9-amino-acid lipopeptide thanamycin  gene clusters, including the two previously described NRPS clusters for thanamycin and the 2-amino acid antibacterial lipopeptide brabantamide. Here we show that thanamycin also has activity against an array of other fungi and that brabantamide A exhibits anti-oomycete activity and affects phospholipases of the late blight pathogen Phytophthora infestans. Most notably, mass spectrometry led to the discovery of a third lipopeptide, designated thanapeptin, with a 22-amino-acid peptide moiety. Seven structural variants of thanapeptin were found with varying degrees of activity against P. infestans. Of the remaining four NRPS clusters, one was predicted to encode for yet another and unknown lipopeptide with a predicted peptide moiety of 8-amino acids. Collectively, these results show an enormous metabolic potential for Pseudomonas sp. SH-C52, with at least three structurally diverse lipopeptides, each with a different antimicrobial activity spectrum.The plant microbiome represents an enormous untapped resource for discovering novel genes and bioactive compounds. Previously, we isolated  Pseudomonas species are ubiquitous in aquatic and terrestrial habitats, and are intensively studied for their abilities to promote plant growth and to suppress plant pathogens , a glycosyltransferase (braA) and a specific FAD-dependent Baeyer–Villiger monooxygenase gene (braC). Biosynthesis of brabantamides is complex: a linear di-peptide is formed by BraB after which the sugar moiety is attached by the glycosyltransferase BraA. This glycosylated di-peptide is subsequently rearranged by the brabantamide-specific monooxygenase BraC  or in King's medium B  agar plates or in LB broth. When needed, growth media were supplemented with 25 μg/ml gentamycin 100 μg/ml kanamycin, and/or 25 μg/ml tetracycline. All bacteria were grown at 25°C.The bacterial strains Pseudomonas sp. SH-C52 for mutants that showed loss of or reduced activity against R. solani  to have a very minor reduction in the growth-inhibitory activity against R. solani (data not shown). The site-directed mutants in the thanamycin gene cluster KO25 and KO26 were obtained previously and were designated in this manuscript as dThaB and dThaC2, respectively. All fungi and oomycetes used in this study were cultured on Potato Dextrose Agar , except for Phytophthora infestans strain 88069 and Phytophthora capsici LT3239 which were cultured on Rye Sucrose Medium and on V8 medium  according to their protocols. For this purpose, two libraries with insert sizes of 0.5 and ~2 kb were constructed and sequenced. In order to ensure the accuracy of follow-up analysis, several steps were performed to filter the raw data: removal of reads with a certain proportion of Ns' bases or low complexity reads (10% as default); removal of reads with a certain proportion of low quality (≤Q20) bases (40 bases as default); removal of adapter contamination (15 bp overlap between adapter and reads as default); removal of contamination due to duplication. For reads with low sequence quality additional processing was performed, i.e., removal of reads with significant poly-A structure, and removal of reads with a k-mer frequency of 1. During the processing 16% of the original read data was eliminated. Short reads were assembled into genomic sequences using SOAPdenovo, a BGI developed assembler  .Sequence information on the thanamycin gene cluster from strain SH-C52  and Pseudomonas mandelii JR-1 (NZ_CP005960) these analyses were performed online at the NCBI database. In general, genes were considered to be present when more than 65% identity with the reference sequence(s), and more than 85% of coverage was observed. In addition to the BLAST analysis, secretion systems were identified by screening gene annotations.Genome comparisons were performed by use of nucleotide BLAST (BLASTN) in BioEdit (Ibis Biosciences). Genes identified by Loper et al.  in speciP. fluorescens group as reported by Redondo-Nieto et al. (Pseudomonas corrugata CFBP5454 (ATKI00000000) and P. mediterranea CFBP5447 (AUPB00000000). Phylogenetic trees were built by a Composition Vector approach using the web server CVTree with a k-value of 6 .A whole-genome phylogenetic analysis was performed with the genomes of species of the o et al. , with thFor the identification of secondary metabolite gene clusters the genome sequence was analyzed by both the on-line analysis programs NP.searcher -domain predicted for the thanapeptin gene cluster was also subjected to phylogenetic analysis (as described above), with C1-domains of other NRPS gene clusters, to compare the thanapeptin C1-domain to cyclic and non-cyclic (lipo)peptides NRPSs  at the periphery of 1/5th strength PDA (pH 7.0) plates. After incubation for 2 days at 25°C, an agar plug (4-mm-diameter) from a freshly grown plate with the target organism was transferred to the center of the 1/5th PDA plate. Inhibition of radial growth of the fungus or the oomycete was monitored from 2 days after incubation up to 2 weeks after incubation, depending on the growth speed of the target organism. For in vitro bacteria and yeast inhibition assays, bacteria and yeast cells were included, after autoclaving and cooling to 45°C, into the 1/5th PDA medium at a concentration of ~105 cfu/ml. After solidifying and sufficient drying of the plates, SH-C52 and its derivative strains were, using an overnight culture in KB, spot-inoculated (3 μl) at the periphery of the bacteria or yeast containing plates.For P. infestans, individual P. infestans mycelial plugs were transferred to a 24-wells plate (Greiner Bio-One) with each well containing 2 ml rye sucrose broth . Brabantamide A treatments were performed simultaneously with the labeling or for 15 min after the labeling. All treatments contained a final concentration of 1% (v/v) DMSO and 0.1% (v/v) of n-butanol. The latter was well below phospholipid metabolism stimulatory concentrations  and two glass beads (Ø = 3 mm) were added and the samples were again frozen in liquid nitrogen. Samples were thawed and thereafter vigorously shaken for 30 min. Samples were further treated and analyzed as described previously  running at a flow rate of 0.8–2.5 μl/min. The droplet size using this configuration was ~200 μm in diameter. MALDI imaging was performed according to Liu et al.  ISP2 agar plates . Streak-inoculated plates were grown at 28°C for 36–44 h. Thanapeptin was extracted by scraping cells from the plates into solvent (60% acetonitrile/40% water/0.1% formic acid). Extractions were performed by shaking for 1 h. The cell extract was transferred to 50-ml centrifuge tubes and cells were spun down. The extraction was performed on the same cells for a total of 3 times. The supernatant was dried by rotovap and the pellet was retrieved by rinsing three times of 6 ml of the extraction solvent. The extraction fluid was collected in a centrifuge tube and spun down again. Subsequently, the supernatant was dried by rotovap again using a small 20-ml scintillation vial until dry. The vial was then rinsed by three washings with 400 μl of extraction solvent and placed in a 1.5 ml centrifuge tube and centrifuged at 14,000 rpm. The supernatant was injected in the HPLC containing a C18 4.6 × 150 mm 5 μm analytical column with a 250 μl sample loop. Mobile phase A was 100% water with 0.1% formic acid. Mobile phase B was 100% acetonitrile with 0.1% formic acid. The flow rate was set to 1.0 ml/min and a gradient elution was used with 15% mobile phase B ramped to 95% mobile phase B over 45 min. The eluent was collected using a fraction collector set to collect 1 ml fractions. Fractions were checked for purity and the right mass using MALDI (Bruker). Purified thanapeptin and its derivatives were subsequently used in antimicrobial assays, and assayed by MS analysis by a Thermo LTQ-FT-ICR MS capable of collision-induced dissociation.For antimicrobial assays and detailed MS analysis of the thanapeptin derivatives, strain Rhodoturula pilimanae, plates containing the target strain were used. Bacteria and yeast were added at a concentration of ~105 cfu/ml to the 1/5th PDA medium. After sufficient drying of the plate, filter discs with the test compounds at indicated concentrations were added to the plate. Plates were incubated at 25°C and growth was monitored for 4 days. Activity against fungal and oomycete strains was tested both in liquid and on plate. For plate assays, thanapeptin derivatives at a concentration of 50 μM were added directly at the border of a 1/5th PDA, after which an agar plug (4 mm diameter) from a freshly grown plate with the target fungal or oomycete strain was positioned in the center of the plate. Hyphal growth was monitored from 2 days after incubation up to 2 weeks after incubation, dependent on the growth speed. Testing of compound activity against fungal and oomycete strains in liquid culture was performed by addition of the compound to 1 ml of liquid 1/5th PDB in a 12 wells culture plate (Greiner Bio-One), in which an agar plug (4 mm diameter) from a freshly grown plate with the target organism was submerged. Growth experiments were incubated at 25°C, except for P. infestans, which was cultured at 18°C.Compounds were tested in interaction with target organisms by different techniques. To test activity against bacteria and the yeast Pseudomonas sp. SH-C52 is ~6.3 Mb with a GC content of 61.0%. The genome was assembled in 596 contigs and 25 scaffolds, with an estimated coverage of 99.2%. For annotation, 384 contigs of at least 100-bp and covering 6.3 MB were used. In total, 5579 ORFs were annotated with 5523 CDSs and 56 tRNAs , levan sucrase (7 strains) and L-tryptophan  are present in multiple strains of the P. fluorescens clade, but were not detected in the SH-C52 genome  corrugatin to its putative gene cluster are still lacking.To predict putative secondary metabolites, such as antibiotics, siderophores and other NRPS genes, the genome sequence was first screened by BLAST for ORFs of known compounds produced by other species and strains of the P. fluorescens strains Q8r1-96 and Q2-87. The second insect toxin gene cluster is similar to that of Tcc4 toxin  flanked by cinA and mutS, typical for prophages present in other Pseudomonas genomes  corrugatin gene cluster Figure , and bramgoA-like gene clusters, encoding only one adenylation domain. MgoA or MgoA-regulated compound(s) were proposed to regulate the expression of pathogenicity factors in P. entomophila and P. syringae  found in SH-C52 is predicted to code for an NRPS that produces an 8-amino-acid lipopeptide, with no significant similarity to other known compounds in general and dedicated databases such as NCBI and the Norine peptide database. This gene cluster appears to be unique for SH-C52, as it was not found in other genomes. The last NRPS gene cluster identified in SH-C52 shows similarity to Rhizoctonia solani . Also cormycin has been shown to be active against B. megaterium and the yeast Rhodotorula pilimanae .Based on mutant analysis, the thanamycin gene cluster was shown to be important for the growth-inhibitory activity of strain SH-C52 against i Figure  and the P. corrugata . However, overnight exposure of P. infestans mycelium to brabantamide A led to an increase in the phosphatidylbutanol (PtdBut) levels. This already occurred at a concentration of 1 μM brabantamide A, the lowest concentration tested -domain, a so-called C1 starter domain (data not shown), suggesting N-acylation of the first amino acid in the peptide moiety. The three NRPS genes of the thanapeptin gene cluster encode nine, four and nine adenylation (A)-domains, respectively. The prediction of the 22 amino acids activated by these A-domains is shown in Figure P. corrugata CFBP5454 draft genome sequence shows that the corpeptin gene cluster sequence is incomplete and scattered over contigs. Nevertheless, for the annotated parts, protein identities with the SH-C52 NRPSs ThpA and ThpC ranged between 75 and 95%, whereas for ThpB identities ranged between 60 and 80%.The thanapeptin gene cluster consists of three NRPS genes. BLAST analysis of the protein sequences showed similarity with the NRPSs for the production of the LP syringopeptin of in silico predicted amino acid sequence for thanapeptin. In addition, tandem MS data provided evidence for cyclization, and resolved the identity of the lipid moiety of thanapeptin . Subsequently, the activity of the derivative with a mass of 2096 Da was tested at different concentrations against the three oomycete pathogens in a liquid broth. For P. infestans , which is in line with results observed previously for syringopeptin and corpeptin (Vassilev et al., The role of the thanapeptin gene cluster in the activity of SH-C52 against fungi, oomycetes and bacteria was studied by comparing wild-type strain SH-C52 and two independent mutants, each with a single transposon insertion in the thanapeptin NRPS gene cluster. The two mutants lost their antagonistic activity against oomycete pathogens, whereas they still had activity against fungi Figure  and bacta Figure . In addis Figure , a clears Figure . No appaPseudomonas sp. SH-C52 was initially studied for its role in the natural soil suppressiveness against the fungal pathogen R. solani (Mendes et al., Pseudomonas sp. SH-C52 can be placed within subgroup I of P. fluorescens species (Redondo-Nieto et al., P. corrugata and P mediterranea. P. corrugata strains have been reported for biological control, for bioremediation and for the production of a range of biomolecules. In contrast, P. corrugata strains have also been reported as plant pathogenic bacteria (Catara, P. corrugata CFBP5454 was isolated because of its pathogenic properties (Licciardello et al., Pseudomonas sp. SH-C52 was isolated because of its plant-beneficial and antagonistic properties (Mendes et al., P. corrugata, regulation of cormycin and corpeptin production is under the control of an N-acyl-homoserine-lactone quorum sensing system (Licciardello et al.,  Catara, . Indeed,Pseudomonas sp. SH-C52 produces a third LP with antimicrobial activity. Previously, brabantamides were shown to be active against specific Gram-positive bacteria (Reder-Christ et al., Pseudomonas sp. SH-C52 a putative fourth LP-NRPS gene cluster is present, with a yet unknown structure and function. Also an mgoA-like NRPS gene was identified in the genome of SH-C52. Although the mgo-operon was shown to be essential for mangotoxin production in mango-pathogenic P. syringae strains (Arrebola et al., mbo-operon (Carrion et al., P. entomophila, however, no pathogenicity factor or compound has yet been linked to this phenotype (Vallet-Gely et al., mgo-operons are not only present in pathogenic bacteria, but are widespread among Pseudomonas species, including species from the P. fluorescens clade (Loper et al., mgo-operon, has not been deduced, the function of the mgo-operon remains an intriguing question. The Mgo-product may act as a Pseudomonas specific switch for secondary metabolite production or as a genus- or species-specific regulator involved in different functions.In addition to thanamycin and thanapeptin, Pseudomonas sp. SH-C52 has the ability to produce at least three and likely four structurally different LPs, each with structural variants. LPs have diverse natural functions in motility, biofilm formation, antibiosis, defense or virulence (Raaijmakers and Mazzola, Pseudomonas CMR12a, i.e., sessilin and orfamide that are produced together but involved in respectively biofilm formation and swarming. Regulation takes place by the fact that sessilin hampers the release of orfamide by co-precipitating with orfamide (D'Aes et al., Pseudomonas SH-C52 (Figure in vitro, but it remains to be studied if these LPs also have antimicrobial activity in vivo. In this respect, future studies will focus on the identification of regulatory pathways and identification of specific extracellular cues that trigger the production of the structurally diverse LPs in SH-C52 in interactions with diverse organisms encountered in the rhizosphere environment.The production of secondary metabolites often comes at the expense of primary metabolism (Ruiz et al., in silico genome analysis and drafted the manuscript. HM performed growth inhibition assays with P. infestans and experiments to test phospholipase activity. JW designed chemical experiments, helped in chemical analysis, and performed the chemistry and analysis on the thanapeptin structure, ED assisted in microbiological and molecular experiments. RM was involved in acquiring and analysing genome information. YS and HG purified brabantamide A for phospholipase and antimicrobial assays. PD designed chemical experiments and was involved in chemical imaging. JR supervised the work and was involved in the experimental design. All authors contributed to the writing of the manuscript and approved submission.MV designed and performed microbiological, molecular and chemical experiments, performed The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Pneumonia is the major cause of sepsis, responsible for almost one-half of all sources of infection . Sepsis Klebsiella pneumoniae as described in [9 colony-forming units to achieve a mortality rate of ~50% by 48 hours. Blood, lung, heart, liver, spleen, kidney and brain were aseptically harvested, homogenized and plated on Müeller-Hinton agar dishes, for 18 hours at 37°C. Plasma and the tissues homogenates were assayed for interleukin-6 (IL-6) and interleukin-1β (IL-1β) using commercially available enzyme-linked immunosorbent assay kits according to the manufacturer's recommendations .Pneumosepsis was induced by ribed in . The numK. pneumoniae evolved to systemic spreading of bacteria to all organs, mostly the liver and kidney. Surprisingly, bacteria were found as early as 30 minutes in vital organs such as the brain and heart. The infection in the organs rose steadily up to 24 to 48 hours. Significant increases in IL-6 and IL-1β were found in the plasma, 24 hours after infection. However, cytokine levels in the organs were as high as fivefold the plasma levels.Lung inoculation with Our data show that the early bacterial dissemination may be important for the onset of organ inflammation. Assuming that the higher organ cytokine level is a marker of an ongoing inflammation, this may explain organ dysfunction during sepsis. Thus, our study suggests that systemic (meaning blood) parameters may not reflect the severity of inflammation/dysfunction in target organs, and that might be the determinant to sepsis outcome.


Adrenomedullin (ADM), a circulating vasodilatory peptide, plays an important role in the development of sepsis-associated hemodynamic and microcirculatory disorders. While administration of exogenous ADM had beneficial effects in several septic animal models, elevated ADM concentrations are associated with a bad outcome. This prompted us to test the effect of various anti-ADM antibodies in a cecal ligation and puncture (CLP) mouse model.in vitro and their ability to reduce mortality in a CLP mouse model were assessed.To gain new potential compounds for the treatment or prevention of septic shock we followed an alternative strategy to influence the ADM system: High-affinity anti-ADM antibodies with different epitope specificities were developed and their antagonist activity p = 0.0004), whereas other antibodies with similar affinities but different epitope specificities were much less potent. The efficacious antibody, in contrast to an anti-C-terminal antibody, only partially inhibited ADM agonist activity in vitro. Healthy mice were not negatively affected by the N-terminal antibody.An anti-ADM antibody directed against the N-terminus substantially increased the survival of mice in a CLP model (HR = 0.077 (CI = 0.0189 to 0.315), An anti-N-terminal ADM antibody, as opposed to antibodies with other epitope specificities, strongly reduces mortality in CLP mice.The online version of this article (doi:10.1186/2197-425X-1-3) contains supplementary material, which is available to authorized users. Despite advances in supportive care, each year 750,000 people develop sepsis and 225,000 die in the USA alone, and the incidence of sepsis is rising at rates between 1.5% and 8% per year . Sepsis The peptide adrenomedullin (ADM) has been implicated in the development of septic shock: It is important in initiating the hyperdynamic response during the early stage of sepsis, and the reduced vascular responsiveness to ADM is associated with the transition from the hyperdynamic phase to the hypodynamic phase during the progression of sepsis –14.ADM comprises 52 amino acids and is secreted into the blood stream from a variety of tissues –17. ADM Apparently, in contrast to the reported beneficial effects of ADM in sepsis, ADM has been categorized as a cardiac depressant factor in sepsis: ADM 22-52), an ADM receptor antagonist, improved the contractility of myocytes isolated from lipopolysaccharide (LPS)-treated rats. In addition, an anti-ADM antiserum improved the survival of LPS-treated rats [-52, an APlasma levels of circulating ADM are relatively stable throughout various clinical conditions . The str2 fragments were generated by standard procedures. For antibody nomenclature, see Table Antipeptide antibodies were generated and selected by standard procedures : Peptides were synthesized, as listed in Table To determine the affinity of the antibodies to human and mouse ADM, respectively, the association and dissociation kinetics of ADM binding to immobilized antibody was determined by means of label-free surface plasmon resonance using a Biacore 2000 system . Reversible immobilization of the antibodies was performed using an anti-mouse Fc antibody covalently coupled in high density to a CM5 sensor surface according to the manufacturer's instructions . Antibody binding kinetics were determined by standard procedures, and affinity constants were calculated .4, 124 mM NaCl, 25 mM HEPES, 13.3 mM glucose, 1.25 mM KH2PO4, 1.45 mM CaCl2, 0.5 g/L BSA). Dose-response curves were obtained in parallel with the reference agonists human ADM and mouse ADM. After addition of the lysis buffer, cAMP was measured with an HTRF kit according to the instructions of the manufacturer . The dose-dependent antagonist activity of anti-ADM antibodies was determined in the presence of human ADM or mouse ADM, respectively, either one at a predetermined concentration yielding 80% of the maximal stimulation obtainable for the cAMP response: 6 μL of the reference agonist (human (5.63 nM) or mouse (0.67 nM) ADM) was mixed with 6 μL of the test samples at different antibody dilutions of 100, 20, 4, 0.8, and 0.16 μg/mL. After incubation for 60 min at room temperature, 12 μL of cells  were added. The plates were incubated for 30 min at room temperature, and cAMP was measured as described above. Antagonist activity of anti-ADM antibodies was calculated as percentage of inhibition relative to the maximal inhibition obtainable with the reference antagonist peptide ADM (22-52).CHO-K1 cells expressing recombinant ADM receptor  grown in media without antibiotic prior to the test were detached by gentle flushing with PBS-EDTA (5 mM EDTA), recovered by centrifugation, and resuspended in assay buffer  were used for the study . Peritonitis had been surgically induced under light isoflurane anesthesia. Incisions were made into the left upper quadrant of the peritoneal cavity . The cecum was exposed and a tight ligature was placed around the cecum with sutures distal to the insertion of the small bowel. One puncture wound was made with a 24-gauge needle into the cecum, and small amounts of cecal contents were expressed through the wound. The cecum was replaced into the peritoneal cavity and the laparotomy site was closed. Finally, the animals were returned to their cages with free access to food and water. Five hundred microliters of saline was given s.c. as fluid replacement.2 fragments of NT-M were tested analogously.Using this CLP model, three anti-mouse ADM antibodies  were tested for their ability to modify the 14-day mortality rate in comparison to vehicle (PBS buffer) and control compound (unspecific IgG). Ten animals per compound were used. Compounds were applied i.v. immediately prior to surgery . Mice were monitored twice daily. In a follow-up experiment, Fab- and F(ab)Twelve- to fifteen-week-old male C57Bl/6 mice  were used for the study. Six mice were treated with NT-M anti-ADM antibody . As control, six mice were treated with PBS (10 μL/g body weight). Survival and physical condition were monitored for 14 days.For all animal experiments described in this manuscript, approval was obtained according to the requirements of the respective German law (Tierschutzgesetz (TierSchG)) from the Niedersächsisches Landesamt für Verbraucherschutz und Lebensmittelsicherheit (LAVES). Our study has been purely experimental and did not involve any human samples.t test. For survival analysis, Kaplan-Meier curves were generated and log-rank test for significance was performed. GraphPad Prism 4 software  was used.Statistical significance was calculated by Student's -9 to 1.6 × 10-8 M  and human ADM  were developed Figure , Table 1The agonist and antagonist activities of the antibodies were tested in an established ADM bioassay system employing a CHO cell line overexpressing the human recombinant ADM receptor (CRLR + RAMP3) with a cAMP readout. None of the antibodies exhibited agonist activity in the bioassay (data not shown). The antibodies showed different dose-dependent antagonist activity profiles Figure , Table 1in vivo should by far exceed on a molar basis the expected concentrations of endogenous plasma ADM. In the control groups (vehicle buffer or unspecific control antibody), most of the animals had died already on day 1 , p = 0.0215; CT-M vs. vehicle: HR = 0.182 (CI = 0.766 to 0.043), p = 0.0202; CT-M vs. control: HR = 0.1796 (CI = 0.044-0.733), p = 0.0167). A strong and sustained improvement of survival was obtained with the antibody against the N-terminus of ADM: 50% of the animals treated with this antibody survived the CLP procedure over the observation period of 14 days , p = 0.0004). The improvement of survival for the antibody against the N-terminus of ADM was significantly higher than for the anti-midregion and the anti-C-terminal antibody (NT-M vs. MR-M: HR = 0.212 (CI = 0.712 to 0.063), p = 0.0122; NT-M vs. CT-M: HR = 0.285 (CI = 0.0823 to 0.979), p = 0.0462). Similar results were obtained in a follow-up experiment with Fab and F(ab)2 fragments of the antibody against the N-terminus of ADM 2: HR = 0.298 (CI = 0.044 to 0.354), p = 0.0025). When the antibody against the N-terminus of ADM was administered to healthy mice even at a 15-fold-higher concentration than that in the CLP experiment, all animals survived and the physical condition of the animals was not impaired compared to control animals (data not shown).The anti-mouse ADM antibodies were tested in a CLP sepsis mouse model for their ability to reduce mortality. The observation period was 14 days. The doses of antibodies were chosen so that concentrations 1 Figure . The antWhen the antibody against the N-terminus of ADM was administered to healthy mice even at a 15-fold higher concentration than in the CLP experiment, all animals survived and the physical condition of the animals was not impaired compared to control animals (data not shown).In this study, we report that an antibody directed against the N-terminus of ADM substantially reduces mortality in a septic animal model, whereas antibodies against other regions of ADM are far less efficacious. Anti-N-terminal antibodies only partially inhibit ADM agonist function in a bioassay.It has been known that plasma ADM levels strongly increase with the severity of sepsis, severe sepsis, and septic shock, and that elevated levels are highly prognostic for fatal outcome –26. ThisIn vitro experiments suggest that ADM has negative inotropic effects and cause impairment of cardiac function in sepsis: The contractility of cardiac myocytes isolated from LPS-treated rats was reduced compared to that in control animals, and addition of ADM (22-52), an ADM receptor antagonist peptide, reversed this effect [ADM might not be beneficial under all circumstances in the progression of sepsis: n of ADM -52, an An of ADM -52, an As effect . The ant2 fragments thereof, which substantially reduced mortality in a mouse CLP model. In contrast, an anti-C-terminal antibody did not strongly improve survival in this animal model. Thus, promotion of survival apparently requires on one hand a certain minimum level of functional ADM and on the other hand the reduction of excessively high concentrations of ADM. It appears paradoxical that both supplementation with ADM, as shown previously [in vivo half-life of only 22 min [We set out to systematically develop and characterize monoclonal antibodies directed against various epitopes of ADM and investigate their potential utility as drugs to reduce sepsis-associated mortality in a mouse CLP model. Antibodies against the C-terminal moiety of ADM were capable of fully inhibiting the ADM-induced cAMP response in an ADM receptor bioassay. This could have been expected as ADM (22-52) is known to be an effective ADM receptor antagonist. Less expected was our finding that antibodies against the N-terminal moiety of ADM inhibited ADM agonist activity maximally by only around 25%. The level of inhibition did not change over almost three orders of magnitude of antibody concentration tested. It was exactly such an anti-N-terminal antibody as well as Fab and F(ab)eviously , 18–21, y 22 min , a relaty 22 min , and they 22 min , and thiIn conclusion, we have shown in an established septic mouse model a strongly beneficial effect on survival by use of an anti-N-terminal ADM antibody. The effect is due to its epitope specificity and is associated with partial inhibition of ADM agonist function. The results of this pilot study are a basis to further develop an alternative, advantageous way for drug development modulating the ADM pathway to finally reduce mortality during the progression of sepsis, which continues to be a strong unmet clinical need.The following are the key messages derived from this study:in vitro depending on their epitope specificity. Anti-ADM antibodies have different antagonist activity  Antagonist activity is strong for anti-C-terminal antibodies, medium for anti-midregional antibodies and weak for anti-N-terminal antibodies. Anti-ADM antibodies are significantly but differently efficacious in reducing mortality in CLP-treated mice depending on their epitope specificity. The anti-N-terminal antibody reduces mortality substantially, whereas antibodies against other regions of ADM are less potent.


E-cadherin, a transmembrane adhesion molecule, and phosphatase of regenerating liver 3 (PRL-3) protein, a member of the family of tyrosine phosphatases, seem to be responsible for cancer cell migration. Therefore, the study objective was to determine a correlation between PRL-3 and E-cadherin, to assess their expression in neoplastic tissue and normal mucosa of the stomach, to analyze their effect on cancer advancement, and to evaluate their potential as prognostic markers in gastric cancer. The expressions of PRL-3 and E-cadherin were assessed immunohistochemically in 71 patients with gastric cancer. Positive expression of PRL-3 was observed in 42.2 % of gastric cancer cases, whereas E-cadherin expression was abnormal in 38 % of cases. The study revealed that the positive PRL-3 expression and abnormal E-cadherin expression were associated with mucinous gastric carcinoma and lymph node involvement. The former was also related to the infiltrating type of tumor and abnormal E-cadherin expression. The expression of PRL-3, but not of E-cadherin, was associated with shorter survival of patients. PRL-3 and E-cadherin exhibit interactions in gastric cancer and are involved in the formation of lymph node metastases. The PRL-3 protein can be an independent predictive factor of overall survival in gastric cancer patients. The phosphatase of regenerating liver 3 (PRL-3) protein belongs to the family of tyrosine phosphatases, with a unique COOH-terminal prenylation motif, and it is thus involved in a major reaction for the cell, i.e., dephosphorylation of tyrosine residues deactivating enzymes. Although its physiological role is poorly investigated, literature data suggest that PRL-3 takes part in neoformation, i.e., in migration, metastasizing, and angiogenesis , 2. HoweE-cadherin is a transmembrane protein, which, in normal epithelium, is responsible for intracellular interactions. Its cytoplasmic domain interacts with β-catenin or γ-catenin. The complex that is formed binds α-catenin, which directly affects the cytoskeleton , 12. In Considering the similar role of both proteins in the migration of cancer cells and EMT, the objective of the current study was to determine the correlation between the PRL-3 protein and E-cadherin, to assess their expression in cancer tissue and in normal gastric mucosa, as well as to investigate their effect on tumor stage. Also, the prognostic potential of both proteins was assessed in gastric cancer.Helicobacter pylori infection was assessed in Giemsa-stained preparations.The study involved 71 patients with gastric cancer treated surgically in the Second Department of General Surgery and Gastroenterology in the years 2005–2010. As a control, healthy gastric mucosa was collected from a stomach fragment removed during therapeutic surgery. The postoperative material was fixed in buffered and paraffin-embedded formalin. From paraffin blocks, 4-μm sections were cut off and stained with hematoxylin-eosin (H + E). Routine histopathological analysis included determination of tumor histological type, malignancy grade (G), anatomoclinical stage (pT), and lymph node metastases. Gastric cancers were also divided according to Lauren’s classification , Goseki’Tissue blocks were cut using a microtome into 4-μm-thick sections on silanized glasses. The sections were deparaffinized in xylenes and hydrated in alcohols. In order to exhibit antigen, the tissue sections were heated in a microwave for 15 min in citrate buffer (pH = 6.0). Then, they were incubated with 0.5 % hydrogen peroxide in methanol to block endogenous peroxidase and, next, with mouse anti-PRL-3 antibody  overnight at 4 °C and with mouse monoclonal anti-E-cadherin antibody (clone 36B5). Following streptavidin-biotin reaction , the antigen-antibody complex was visualized by application of chromogen 3,3′-diaminobenzidine . The expressions of the proteins were assessed using the semiquantitative method, which defined PRL-3 expression as positive when the reaction was visible in more than 5 % of cancer cells and negative when there was no reaction or it was present in <5 % of cells. For E-cadherin, the reaction was normal when the membranous expression of the protein was present in >30 % of cells; it was abnormal when negative or when membranous expression was present in <30 % of cells or in the case of cytoplasmic expression. Positive reaction was calculated in at least 500 cancer cells in each tissue section using a light microscope (×400).χ2 test. Log-rank test which is according to Kaplan-Meier survival analysis approach was employed to compare the overall survival rate of patients. A Cox proportional hazard model was used for univariate and multivariate analyses. A p value of <0.05 was considered statistically significant. The statistics was performed using STATISTICA 10 (Poland).The statistical analysis was based on Fisher’s test and Normal gastric mucosa did not show PRL-3 expression, whereas E-cadherin was present on the cytoplasmic membrane of most glandular cells. The neoplastic tissue had a positive expression of PRL-3 in 42.2 % 30/71) of gastric cancer cases, whereas abnormal expression of E-cadherin was observed in 38 % (27/71) of cases . Positive expression of PRL-3 was also related with the infiltrating type of tumor in Borrmann’s classification (p = 0.037). Patients with local lymph node involvement also had increased PRL-3 and abnormal E-cadherin expression in primary tumor (p < 0.001). Moreover, positive PRL expression was associated with abnormal expression of E-cadherin (p = 0.007) , malignancy grade, tumor type according to Goseki and Lauren, and the presence of p = 0.510). However, the postoperative time of overall survival in patients with positive PRL-3 expression was markedly shorter (p = 0.023) . Malignancy grade, tumor type in Lauren’s classification, and positive PRL-3 expression were independent predictive factors of overall survival for these patients  , which indicates that they may interact.EMT is a physiological process of growing embryos during which epithelial cells are transformed into mesenchymal cells. In a similar way, cancer cells of epithelial origin change into mobile mesenchymal cells. Cell junctions are lost, and cancer cells in this form are able to migrate to remote parts of the body. After being released from the primary tumor, they invade the surrounding tissues, penetrate lymphatic or blood vessels, and migrate through the vascular wall, where they eventually settle, proliferate, and induce angiogenesis. There, they become transformed into epithelial cells and adhere tightly to one another to form a metastatic tumor . The abiWe also compared the relationship of PRL-3 and E-cadherin with clinicopathological parameters in gastric cancer. We revealed a correlation of positive PRL-3 and abnormal E-cadherin with mucinous type of gastric cancer. Similar observations have been reported by other authors, indicating a relationship between these proteins and signet ring cell-type carcinoma , 22. It We also analyzed whether the proteins could be prognostic factors in gastric cancer. Only the positive expression of PRL-3 was found to correlate with shorter survival of patients. We also demonstrated that PRL-3 was an independent prognostic factor and could be used as a therapeutic target in this cancer. Ooki et al.  have addIn conclusion, our study found a significant correlation between PRL-3 protein and E-cadherin. However, it is the PRL-3 protein which seems to be more important in the growth of gastric cancer.


To assess the non-specific effect of Bacillus Calmette-Guérin (BCG) vaccination at birth on psychomotor development.This is a pre-specified secondary outcome from a randomised, clinical trial.Maternity units and paediatric wards at three university hospitals in Denmark.Children born at gestational age (GA) 32 weeks and above. All women planning to give birth at the three sites were invited during the recruitment period. Out of 4262 randomised children, 144 were premature (GA < 37 weeks). There were 2129 children (71 premature) randomised to BCG and 2133 randomised (73 premature) to the control group.BCG vaccination 0.05 ml was given intradermally in the upper left arm at the hospital within seven days of birth. Children in the control group did not receive any intervention. Parents were not blinded to allocation.Psychomotor development measured using Ages and Stages Questionnaire (ASQ) completed by the parents at 12 months. Additionally, parents of premature children  completed an ASQ at 6 and 22 months. Developmental assessment was available for 3453/4262 (81%).d = -0.015 (-0.082 to 0.052). The mean difference in ASQ score for premature children at 22 months was -7.8 points , d = -0.23 (-0.62 to 0.15).The mean difference in ASQ score at 12 months adjusted for age and prematurity was -0.7 points , p = 0.67, corresponding to an effect size of Cohen’s A negative non-specific effect of BCG vaccination at birth on psychomotor development was excluded in term children.NCT01694108ClinicalTrials.gov  M. tuberculosis and progression to tuberculous disease in children[The Bacillus Calmette-Guérin vaccine (BCG) against tuberculosis is one of the most commonly used vaccines globally. Approximately 100 million children receive it every year. The vaccine provides some protection against both infection with  children. A rando children and seve children–7 have s children among BC children. Child m children.To our knowledge, the possible non-specific effects of BCG on child psychomotor development have not previously been studied. BCG vaccination induces inflammatory Th1-responses and Th2-responses to unrelated pathogens in vaccinated children,13. BCG Therefore, a secondary outcome of The Danish Calmette Study was to aThis study reports on a secondary outcome of The Danish Calmette Study, described in detail elsewhere. Initialwww.clinicaltrials.gov (NCT01694108). The trial was supervised by the Good Clinical Practice Units of the Capital Region and the Region of Southern Denmark. The study was monitored by an independent data and safety monitoring board with three members. All parents gave written informed consent.The study was approved by the Committees on Biomedical Research Ethics (J.no. H-3-2010-087), the Danish Data Protection Board (J.no. 2009-41-4141), and the Danish Medicines Agency (J.no. 2612–4356. EudraCT 2010-021979-85. Protocol 2009–323) and registered at The trial took place at maternity units and paediatric wards at three university hospitals in Denmark. Within seven days of birth, study staff randomised children to receive BCG (Danish SSI strain 1331) in the standard dose of 0.05 mL intra-dermally on the left shoulder or to a no-intervention control group. Parents were not blinded to allocation since the local inflammatory reaction after BCG vaccination could not be mimicked. Randomisation was stratified by prematurity  and children were allocated 1:1 in permuting blocks of 2-4-6 using an online system. Exclusion criteria were GA < 32 weeks, birth weight < 1000 g, child critically ill, known immune-deficiency, no Danish-speaking parent and maternal use of immune modulating drugs during pregnancy. Follow-up consisted of telephone interviews and clinical examination at 3 and 13 months as well as hospital admission diagnoses obtained from national registries at 15 months.a priori.The Ages and Stages Questionnaire (ASQ) is a series of validated parent-reported, age-related questionnaires ranging from 2 to 60 months, designed to identify children with developmental delay. Each questionnaire contains 30 items on every-day child activities within five sub-domains . Each itnd edition) translated for previous research use[ASQ has not been published in Danish. We obtained permission from the publisher to use a version  was determined to be able to detect a 20% reduction in number of hospitalisations up to 15 months with a power of 90%. With thresponders if the questionnaire was complete, partial responders if the questionnaire was incomplete and non-responders if the questionnaire was missing.At each follow-up, we grouped parents into d effect size; calculated as the beta-coefficient divided by the square root of the mean residual variance. The primary analysis was a regression model adjusting for prematurity using complete cases only. Secondary outcomes are reported if all questions within the ASQ sub-domain were answered. A primary outcome sensitivity analyses was conducted adjusting for variables that could be associated with missing outcome; i.e. age at follow-up, child sex, study site, gestational age in days, maternal year of birth, maternal education, multiple birth, maternal smoking during pregnancy, older siblings. Due to a data management system error premature children with gestational age of 32 weeks to 33 weeks and 3 days did not receive the ASQ at 6 months. This mechanism of lost to follow-up was interpreted as missing at random since the probability of being missing was not associated with the outcome. To explore this, we used general/multivariate normal linear models estimated by maximum likelihood to examine the associations between BCG and ASQ score across time for premature children taking into account correlation of measurements within children. The model includes a follow-up variable and an interaction between BCG and follow-up allowing separate effects of BCG to be estimated at 6, 12, and 22 months. Unstructured covariance matrices were used to keep variances and correlations unconstrained. The model is often called Multivariate- or Mixed Model Repeated Measures denoted MMRM.Continuous outcomes were analysed using multiple regression models adjusting for child age at follow-up and for prematurity , and raw estimates are presented as well as Cohen’s U-test. Categorical outcomes were analysed using χ2. The significance level was 0.05 and all tests were two-tailed. All analyses reported are intention-to-treat analyses.Slight right-truncation was detected at 12 month follow-up  but the All analyses were performed using STATA 13.1 . The statistical code used is available in Between October 2012 and November 2013, a total of 16 521 pregnant women were invited. We enrolled 4184 mothers and their 4262 newborn children were randomised . BaselinThere were 350/2129 (16.4%) partial responders and non-responders in the BCG group vs. 459/2133 (21.5%) in the control group, p < 0.001. Among partial responders and non-responders, there were significantly more women with basic schooling and non-theoretical training , and more families with siblings .There were 46/144 (32.4%) premature partial responders and non-responders at 6 months, 29/144 (20.1%) at 12 months and 22/144 (15.3%) at 22 months.The mean ASQ score among the 3453 children with complete questionnaires at 12 months was 178.2 points (SD 52.4) in the BCG group vs. 179.9 points (SD 53.6) in the control group. A total of 22 children scored the maximum score of 300.d = -0.015 (-0.082 to 0.052). There were no statistically significant differences in any of the ASQ sub-domains (d = -0.23 (-0.62 to 0.15). The mean differences in premature children at 6 months and 12 months were also negative but not statistically significant and the effect of BCG was not statistically significantly different between premature and term children at 12 months , corresponding to an effect size of -domains . At 22 m2 months . The res2 months .In premature children, the effect of BCG did not differ significantly at 6, 12, and 22 months, p = 0.47. The mean effect of BCG was not significant when combining the three estimates; BCG vs. control: -6.4 points (-15.6 to 2.8), p = 0.17. There is no consistent pattern in which sub-domain scores are affected at 6, 12, and 22 months for premature children . Adjustmd = -0.072 (-0.16 to 0.020) and 2.0 points (-2.4 to 6.4), d = 0.045 (-0.052 to 0.14) for girls. The difference in effect between boys and girls was not significant (p = 0.087 for interaction).In the entire cohort at one year, the mean, age-adjusted BCG effect for boys was -3.2 points (-7.4 to 0.9), In the present study, we report a pre-specified secondary analysis of a randomised, clinical trial, investigating whether BCG vaccination at birth affects the psychomotor development of children. We found no effect of BCG on the psychomotor development at 12 months of age.The study has sufficient power to detect even a small difference in ASQ score among the mature children and thus has a low risk of not detecting a clinically relevant effect. To measure development, we used a well-known and extensively used questionnaire, the ASQ, which is completed by parents and has been shown to have high validity, sensitivity and specificity as a tool for detecting developmental delay. We succStudying potentially negative effects in a trial is only ethically permissible if the expected benefits outweigh the possible side effects. With an expected benefit of a 20% reduction in hospitalisation, we argue that the equipoise required for conducting the trial ethically was presIn order to corroborate or refute the negative effect estimate we found in premature children, we suggest that neurodevelopmental outcomes are assessed, preferably using ASQ, as part of ongoing and future BCG trials for premature children, even if data would perhaps need to be pooled across studies in order to achieve sufficient power. Perhaps especially so in populations with low prevalence of tuberculosis and a low burden of neonatal infections, i.e. a setting in which the non-specific beneficial effect of BCG on mortality is likely to play a minor role and where prevention of tuberculosis is not a priority at the community level. Conversely, in low-resource settings where a beneficial effect of BCG on all-cause mortality has been demonstrated, the findings from this study should not deter BCG vaccination of premature children, who seem to benefit the most.A clinically significant negative non-specific effect of BCG vaccination at birth on psychomotor development at 12 months of age was excluded in children born to term. No clear conclusion on effect in premature children (GA < 37 weeks) was possible in the present study.S1 FigASQ: Ages and stages questionnaire.(TIF)Click here for additional data file.S1 File(DOCX)Click here for additional data file.S2 File(PDF)Click here for additional data file.S3 File(PDF)Click here for additional data file.S4 File(DOCX)Click here for additional data file.S5 File(DOC)Click here for additional data file.S6 File(DOCX)Click here for additional data file.S1 Text(DOCX)Click here for additional data file.S2 Text(DOCX)Click here for additional data file.


Further investigation of the underlying mechanisms revealed that BANCR markedly activated autophagy. Overexpression of BANCR inhibited apoptosis in the IHH-4 cells, whereas inhibition of autophagy stimulated apoptosis in the BANCR-overexpressed cells. BANCR overexpression also increased cell proliferation and the inhibition of autophagy abrogated BANCR overexpression-induced cell proliferation. In addition, the overexpression of BANCR resulted in an increase in the ratio of LC3-II/LC3-I, a marker for autophagy, while the knockdown of BANCR decreased the ratio of LC3-II/LC3-I. These results revealed that BANCR expression levels are upregulated in PTC. Additionally, BANCR increases PTC cell proliferation, which could activate autophagy.Long non-coding RNAs (lncRNAs) are novel regulators in cancer biology. BRAF-activated lncRNA (BANCR) is overexpressed in melanoma and has a potential functional role in melanoma cell migration. However, little is known about the role of BANCR in the development of papillary thyroid carcinoma (PTC). In the present study, BANCR expression was examined in six pairs of PTC and matched adjacent normal tissues. The results revealed that BANCR levels were significantly higher in the PTC tissues and PTC IHH-4 cells compared with the normal controls. Knockdown of BANCR in the IHH-4 cells inhibited proliferation and increased apoptosis of the cells  Papillary thyroid carcinoma (PTC) is the most common type of thyroid cancer, accounting for ~80% of all thyroid cancers. With appropriate treatment, including surgery and radioiodine ablation, the majority of PTCs have excellent prognoses . ExaminiV600EBRAF, which is the most prevalent mutation of the BRAF gene. V600EBRAF mutations are exhibited in 70% of malignant melanomas, 36–53% of papillary thyroid cancers and 5–22% of CRCs  are now recognized as regulators in tumorigenesis and tumor progression ,3. Functs of PTC , but theAutophagy is a lysosome-mediated intracellular catabolic process by which cells remove damaged organelles and long-lived proteins to maintain cellular homeostasis . AutophaThe aims of the present study were to detect the expression levels of BANCR and to investigate the function and molecular mechanisms of BANCR in PTC.In total, six specimens of human PTC and adjacent normal tissues were obtained, with informed consent, from surgeries performed between March and June 2013 at the First Affiliated Hospital of Nanjing Medical University . The protocol used in this study was approved by the hospital’s Protection of Human Ethics Committee. The diagnosis of PTC was histopathologically confirmed. The resected tissue samples were immediately frozen in liquid nitrogen and stored at −80°C until RNA extraction. The human PTC-derived cell line, IHH-4, was provided by Professor Congyou Lu . The IHH-4 cells were routinely cultured at 37°C in RPMI 1640 medium  with 10% fetal bovine serum  and 5% carbon dioxide.−ΔΔCT method was used to determine the relative gene expression levels, using β-actin as the endogenous control to normalize the data. The primers used in this study were as follows: BANCR forward, 5′-ACAGGACTCCATGGCAAACG-3′ and reverse, 5′-ATGAAGAAAGCCTGGTGCAGT-3′; β-actin forward, 5′-AGAAAATCTGGCACCAACC-3′ and reverse, 5′-TAGCACAGCCTGGATAGCAA-3′; LC3 forward, 5′-CCACACCCAAAGTCCTCACT-3′ and reverse, 5′-CAC TGCTGCTTTCCGTAACA-3′. PCR was performed at 95°C for 30 sec, 40 cycles of 95°C for 5 sec, 60°C for 31 sec, and then, for dissociation, at 95°C for 15 sec, 60°C for 1 min and 95°C for 15 sec.Total RNA from tissues and cells was extracted using RNAiso Plus  Co., Ltd., Dalian, China), and reverse transcription (RT) reactions were performed using a PrimeScript RT reagent kit  Co., Ltd.) according to the manufacturer’s instructions. Quantitative PCR reactions were prepared at a final volume of 20 μl using a standard protocol and the SYBR Green PCR kit , and the reactions were performed on the StepOnePlus Real-Time PCR System . Each reaction was performed in triplicate. The 2Recombinant lentiviruses containing short hairpin (sh)RNA-323 , shRNA-540 , human full-length BANCR cDNA (LV-BANCR) and a negative control (LV-NC) were purchased from GenePharma Co., Ltd. . The IHH-4 cells were infected with LV-BANCR-323, LV-BANCR-540, LV-BANCR and LV-NC . The supernatant was removed after 24 h and fresh culture medium was added to the cells. The infection efficiency was confirmed by RT-PCR at 72 h post-infection, and the cells were treated with 2 μg/ml puromycin for 2 weeks.3 cells per well and cultured in the growth medium. The number of cells per well was measured by the absorbance at 450 nm at the indicated time-points, according to the manufacturer’s instructions.Cell proliferation assays were performed using Cell Counting Kit-8 . The cells were plated in triplicate in 96-well plates at ~2×105) were seeded in 6-well plates. After 24 h, the cells were collected and incubated with Annexin V-fluorescein isothiocyanate and 7-amino-actinomycin D  for 15 min in the dark and apoptosis was analyzed using a flow cytometer. The cell cycle was also analyzed subsequent to propidium iodide staining for 30 min.The cells . Medium supplemented with serum was used as a chemotactic agent in the lower chamber. The cells were incubated for 24 h, and those cells that did not migrate through the pores were removed with a cotton swab. The cells on the lower surface of the membrane were stained with crystal violet . The cell numbers were determined by counting the penetrating cells under a microscope  in random fields, with five fields per chamber. Each experiment was performed in triplicate.In total, 4×10Proteins were extracted with radioimmunoprecipitation assay (Beyotime Institute of Biotechnology) and equal amounts of protein were electrophoresed on a 12 or 15% sodium dodecyl sulfate-polyacrylamide gel and subsequently transferred to polyvinylidene fluoride membranes . The membranes were blocked with 5% skimmed milk in Tris-buffered saline containing 0.1% Tween-20 (TBST), at room temperature for 2 h. The membranes were incubated with the rabbit polyclonal LC3-I, LC3-II  and rabbit polyclonal GAPDH  primary antibodies at 4°C overnight. The membranes were then washed three times with TBST and incubated with horseradish peroxidase-conjugated secondary anti-rabbit antibody  at room temperature for 2 h. Following three washes with TBST, the membranes were developed using ECL Plus , and exposed to X-ray film. GAPDH was used as an internal loading control.2-test or one-way analysis of variance, as appropriate. P<0.05 was considered to indicate a statistically significant difference.Data were analyzed using SPSS 19.0 software , and are expressed as the mean ± standard deviation of data from at least three independent experiments. The differences between the groups were analyzed using Student’s t-test, Pearson’s χTo investigate the role of BANCR in PTC development, BANCR RNA expression levels in PTC tissue samples were examined first. The RT-PCR results showed that BANCR expression was significantly higher in five out of six of the tumor tissues compared with the adjacent normal tissues. Additionally, BANCR expression in the PTC IHH-4 cell line was upregulated compared with the mean expression level of the adjacent normal tissues P<0.05; .1 phase , an inhibitor of autophagy. The results revealed that following infection with LV-BANCR, the BANCR expression level was significantly upregulated compared with the LV-NC P<0.05; . The ove P<0.05;  in the B P<0.05; . The RT- P<0.05; .Advances in molecular techniques have led to the identification of a novel type of gene regulators called lncRNA. These lncRNAs are >200 nucleotides and do not code for proteins. However, they can interact with proteins and can likely act as regulators of other genes . Althougin vitro , a senescence-like phenotype and initiates autophagy through the inhibition of mammalian target of rapamycin complex signaling (1 phase; all these effects could be suppressed by 3-MA, an inhibitor of autophagy. These findings suggest that BANCR may increase PTC cell proliferation by activating autophagy.Autophagy is a self-degradative process through which the cytoplasmic materials within the lysosome are degraded. The process acts as a dynamic system that provides the building blocks of a cell and the energy for cellular homeostasis and regeneration . Maddodiignaling . BANCR wTo the best of our knowledge, this is the first study to report that BANCR is highly expressed in PTC and that BANCR is likely to be a useful biomarker of this disease. Additionally, the fact that BANCR increases PTC cell proliferation by activating autophagy adds to our understanding of the molecular mechanisms governing BANCR. Significantly, BANCR could be used as a potential molecular target to treat human PTC.


Arabidospsis thaliana to trace S- and R-IM enantiomer concentrations and examined IM toxicity effects on the root proteome using iTRAQ. Conventional analyses of root carbohydrates, organic acids, and enzyme activities were also performed. We discovered several previously unknown key biochemical pathways targeted by IM in Arabidospsis. 1,322 and 987 proteins were differentially expressed in response to R- and S-IM treatments, respectively. Bioinformatics and physiological analyses suggested that IM reduced the BCAA tissue content not only by strongly suppressing BCAA synthesis but also by increasing BCAA catabolism. IM also affected sugar and starch metabolism, changed the composition of root cell walls, increased citrate production and exudation, and affected the microbial community structure of the rhizosphere. The present study shed new light on the multiple toxicity mechanisms of a selective herbicide on a model plant.Imazethapyr (IM) is a widely used chiral herbicide that inhibits the synthesis of branched-chain amino acids (BCAAs). IM is thought to exert its toxic effects on amino acid synthesis mainly through inhibition of acetolactate synthase activity, but little is known about the potential effects of IM on other key biochemical pathways. Here, we exposed the model plant  In recent years the proportion of herbicide consumption over that of all pesticides has increased rapidly, from 20% in 1960 to 48% in 2005R-IM enantiomer generally being much more toxic than S-IM4et al. demonstrated that IM enantiomers selectively damaged root hair growth and significantly reduced the sloughing of border cells from root tips in maize (Zea mays L.)Oryza sativa) and the model plant Arabidopsis thaliana to trace IM concentrations also caused an imbalance in the antioxidant system and disturbed starch and sugar metabolism in an enantioselective manner5Imazethapyr (IM) is one commonly applied chiral herbicide in rice, soybeans, groundnuts and other crops that selectively control dicot weedsA. thaliana, in response to a 4-day exposure to 20 μg L−1S-IM or R-IM. This approach allows the simultaneous identification and quantitative comparison of peptides by measuring peak intensities of reporter ions in tandem mass spectrometry (MS/MS) spectra. With this technology, we have quantitatively identified, to the best of our knowledge, the most complete proteome in Arabidopsis roots when compared to other studies89R- or S-IM. Bioinformatics analysis showed that the quantifiable proteins were involved in amino acid synthesis and metabolism, the phenylpropanoid pathway, oxidative stress protection, the TCA cycle, as well as sucrose and starch metabolism. Besides iTRAQ analyses, we also evaluated the effect of IM on the rhizosphere microbial community structure, root enzyme activities as well as on the root concentrations of metals, nutrients, carbohydrate and organic acids (OA) in order to better understand IM toxicity mechanisms. The present study unravels several of the complex molecular bases underlying the enantioselective toxic effects of the herbicide IM in a model plant. This new knowledge could be helpful for genetic engineering of herbicide-resistant crops and management of pesticide application in crops.Even though our knowledge of the IM toxicity effects and mechanisms in plants has improved in recent years, the precise cell mechanisms by which IM enantioselectively exerts its toxic effects are yet to be fully understood. Limited information is available on how IM induces changes in the expression of the whole proteome, and on its toxic effects on a wide range of biochemical pathways in a given plant organism. In order to refine our knowledge of IM toxicity mechanisms, we decided to apply a novel high-throughput technology, i.e. mass spectrometry coupled to protein labeling by isobaric tags for the relative and absolute quantitation (iTRAQ), and analyze the proteome of the roots of a model plant species, −1S-IM an R-IM , but R-IM increased the abundance of the large ALS subunit (AT3G48560.1) to 1.3-fold that of the control.Because IM is thought to control weeds primarily by reducing the levels of three BCAAs  through the inhibition of acetolactate synthase (ALS), we first analyzed the proteins associated with amino acid metabolism . SurprisA. thaliana to IM showed that this herbicide decreased the expression of a number of proteins involved in BCAA synthesis  and 3-isopropylmalate dehydrogenase 1 (AT1G31180.1) expression to 1.5- and 2.0-fold that of the control, thereby inhibiting Leu and Ile synthesis. Moreover, S-IM decreased 3-isopropylmalate dehydrogenase 2 (AT1G80560.1) to 1.3-fold that of the control, but it increased ketol-acid reductoisomerase (AT3G58610.3) and BCAA aminotransferase 4 . Again, the R-IM enantiomer was more toxic than the S-IM enantiomer as demonstrated in the proteome of A. thaliana.Furthermore, our proteomic analysis showed that IM stimulated BCAA catabolism . Indeed,R- and S-IM. Among these SUS proteins, SUS4 expression in the root is higher than in other A. thaliana tissues according to Bieniawska et al.R-IM treatment   . More spR-IM , 2-oxoglutarate dehydrogenase (AT5G65750.1), succinyl-CoA ligase [ADP-forming] subunit alpha-1(AT5G08300.1), and malate dehydrogenase  , which is a key enzyme producing acetyl-CoA from citrate during fatty acid synthesis in the chloroplast. In contrast, S-IM increased the expression of ATP-citrate lyase A-3 .The mean pyruvate concentration increased to 1.7-fold that of the control after treatment with R-IM . iTRAQ a56720.1) . MoreoveR-IM treatment, S-IM treatment also increased the abundance of several TCA-related proteins, such as citrate synthase 2 , the 2-oxoglutarate dehydrogenase E1 component (AT3G55410.1) and MDH leading to an increase in oxaloacetate (OA) synthesis. The activities of OA synthesis-related enzymes were also stimulated in response to S-IM (MDH) and R-IM treatment (MDH and PEPC) (S-IM-induced decrease in the expression of succinate dehydrogenase iron-sulfur subunit 1 (AT3G27380.2) should also contribute to a reduction in respiration rate through the TCA cycle compared to that in the presence of R-IM.As observed for nd PEPC) . FurtherR-IM, which should stimulate respiration through the TCA cycle and cause OA synthesis  in the R-IM-treated sample and 6.2 × 108 copies of 16S rDNA/mg F.W. in the S-IM treated sample whereas only 2.3 × 107 copies of 16S rDNA/mg F.W. were measured in the control (We observed the root structure of IM-treated plants (grown in non-sterile conditions) by scanning electron microscopy (SEM). Root cell length decreased and its diameter increased in response to IM exposure, especially after reatment . We also control . We subs control .A. thaliana cultures. The bacterial diversity was similar for all definitions of OTUR-IM, S-IM and control samples (R-IM treatment and was higher in the S-IM treatment than in the control), reaching average values at the unique sequence level of 385.44/485.71 (Chao/ACE index) for R-IM treatment, 304/304.53 (Chao/ACE index) for S-IM treatment and 257.03/261.54 for the control. The S-IM-treated group appeared to be more diverse based on two widely used diversity indices, the Shannon’sR-IM-treated group showed the lowest diversity among the three groups based on the coverage index   . ACLA su(ACLA) 26 should a(ACLA) 26. In respexposure . An incrposure 28, the rooposure 28. The obsIM also affected starch synthesis through the inhibition of several sucrose synthase enzymes . The SUSs are required for defense against biotic and non-biotic stresses, and are thus strongly affected by pathogen interactions and anoxia in several species323335A. thaliana, but also provide evidence that the toxic effect of IM on A. thaliana is related to changes in diversity and abundance of microorganisms in the rhizosphere . Roots are known to excrete secondary metabolites that act as messengers that attract Rhizobium and arbuscular mycorrhizal fungiet al. demonstrated that the secretion of the TCA cycle intermediate L-malic acid from A. thaliana roots is used to recruit the beneficial rhizobacterium Bacillus subtilisA. thaliana roots  and/or direct toxic effect of IM on bacteria in the rhizosphere. Irrespective of the precise mechanism(s) explaining the effect of IM on the bacterial community structure of the rhizosphere, the strong effect of IM on the microorganisms of the rhizosphere revealed in this study is important because it is expected to affect plant nutrition and perhaps indirectly modulate IM toxicity in A. thaliana ecotype Columbia seeds were sterilized with ethanol (75%) and HgCl (0.1%). The sterile seeds were subsequently vernalized at 4 °C for 4 days and transferred onto sponges, which were placed into 1.5 mL Eppendorf tubes from which the bottoms were cut, allowing root growth into the nutrient solution. Murashige and Skoog (MS) nutrient solution was used  and the intial pH was adjusted to 5.6−1R- or S-IM in MS nutrient solution . The plants were grown in a non-sterile growth chamber at 25 °C under a 12 h light/12 h dark cycle at a light intensity of 40 μmol photons m−2 sec−1. The concentration of 20 μg L−1 IM, which was close to the IC50 on the root fresh weight of A. thaliana  equipped with WinRHIZO software (Regent Instruments Corporation) was used to measure several root morphological parameters for each plant, i.e. total root length, surface area, volume, diameter, and number of root tips. Four replicates were used in each treatment, and each replicate consisted of 5 randomly selected seedlings.A. thaliana, control and IM-treated roots of A. thaliana were collected, dried, transferred in acid-washed plastic containers and digested in the microwave with 68% HNO3 for 5 min at 700 W, followed by 10 min at 700–1200 W and 20 min at 1200 W. Subsequently, samples for metal analyses were diluted in Milli-Q water to achieve a final 5% v/v HNO3 concentration prior to analysis by inductively-coupled plasma mass spectrometry .To measure the concentrations of nutritional metals  and other nutritional elements  in roots of A. thaliana was measured according to the procedure detailed in Qian et al.et al.5. The root tissues were stained according to the method of Takahashi et al.The content of glucose, sucrose, and fructose in the roots of et al.We quantified the 16S rDNA abundance per root fresh weight by qRT-PCR according to the method detailed in Qian et al.Metagenomic sequencing was performed according to Bowman A. thaliana roots (approximately 0.4 g fresh weight) were extracted and digested with trypsin (Promega) at an enzyme-to-substrate ratio of 1:50 for 12 h at 37 °C. After iTRAQ (AB Science) labeling, equal amounts of labeled peptides from each group were mixed and resolved into 15 fractions by high performance liquid chromatography (HPLC), followed by Q Exactive mass spectrometry (Thermo Fisher Scientific). The resulting MS/MS data were searched against a Uniprot Arabidopsis protein database with MaxQuant (v1.0.13.13) with 1% FDR . A functional annotation of the quantified proteins was performed with GO (Gene ontology), KEGG (Kyoto encyclopedia of genes and genomes) pathway analysis, functional enrichment analysis and secondary structure analysis. Two technical replicates with tag swapping were also conducted for each biological replicate. Further details on MS/MS raw data database searching, peptide and protein identification and quantification are provided in supplemental materials  was required for protein identification. Two criteria were used for the quantitation of the identified proteins: 1) the median protein ratio was chosen (excluding the outliers); 2) the minimum precursor charge was set to 2 and only unique peptides were used for quantitation. A 1.3-fold up- or down-regulation was chosen to identify significant over- or under-expression, respectively, of proteins in addition to P-value < 0.05.Statistical differences among the mean root contents in polysaccharides, metals, organic acids as well as in root enzyme activity (p < 0.05) were tested with analyses of variance (ANOVA) using the program StatView. The preliminary conditions of ANOVAs  were tested and if the conditions were violated, the data were transformed prior to ANOVAs.How to cite this article: Qian, H. et al. Analyzing Arabidopsis thaliana root proteome provides insights into the molecular bases of enantioselective imazethapyr toxicity. Sci. Rep.5, 11975; doi: 10.1038/srep11975 (2015).


The inherited metabolic disease phenylketonuria (PKU) is characterized by increased concentrations of phenylalanine in the blood and brain, and as a consequence neurotransmitter metabolism, white matter, and synapse functioning are affected. A specific nutrient combination (SNC) has been shown to improve synapse formation, morphology and function. This could become an interesting new nutritional approach for PKU. To assess whether treatment with SNC can affect synapses, we treated PKU mice with SNC or an isocaloric control diet and wild-type (WT) mice with an isocaloric control for 12 weeks, starting at postnatal day 31. Immunostaining for post-synaptic density protein 95 (PSD-95), a post-synaptic density marker, was carried out in the hippocampus, striatum and prefrontal cortex. Compared to WT mice on normal chow without SNC, PKU mice on the isocaloric control showed a significant reduction in PSD-95 expression in the hippocampus, specifically in the granular cell layer of the dentate gyrus, with a similar trend seen in the cornus ammonis 1 (CA1) and cornus ammonis 3 (CA3) pyramidal cell layer. No differences were found in the striatum or prefrontal cortex. PKU mice on a diet supplemented with SNC showed improved expression of PSD-95 in the hippocampus. This study gives the first indication that SNC supplementation has a positive effect on hippocampal synaptic deficits in PKU mice. The primary defect in the inherited metabolic disease phenylketonuria (PKU) is the disrupted phenylalanine (Phe) metabolism, caused by mutations in the gene encoding for the hepatic enzyme phenylalanine hydroxylase, which normally converts Phe to tyrosine. When no dietary Phe restriction is applied, this causes an increase in blood and brain Phe concentration compared to healthy controls. Although many questions still remain to be answered, a clear correlation between Phe concentration in blood and brain and the cognitive symptoms of PKU has been shown ,2,3. IncPhe-induced neuro-morphological changes are reversible, providing a window of opportunity for interventions even after the expression of symptoms due to high Phe exposure ,18. Our enu2  mice and their wild-type  littermates were fed for 12 weeks with different diets. Mice were bred at the University of Groningen, The Netherlands. At the start of the experiment all mice were housed singly. The genotype of the animals was established by quantitative PCR analysis from DNA extracts from tail tissue [In this study, 31-day-old male and female homozygous C57BL/6 Pahl tissue . PKU micl tissue . The difl tissue . This stAfter 12 weeks of dietary treatment, all animals were euthanized via a single intraperitoneal injection of pentobarbital. Blood was collected via heart punction and animals were transcardially perfused with 4% paraformaldehyde ; pH 7.4). Brains were post-fixed for 24 h and subsequently rinsed with 0.01 M PB. After exposing the brains to 30% buffered sucrose, they were snap-frozen with liquid nitrogen and stored at −80 °C.2O2 for 30 min; (2) incubation with 1:1000 monoclonal mouse anti-PSD-95, Millipore, MABN68, 1% normal goat serum (NGS) and 0.5% Triton-X for 2 h in a water bath at 37 °C, 24 h at room temperature and subsequent storage for 48 h at 4 °C; (3) incubation with secondary antibody solution  for 2 h at room temperature (RT); (4) incubation with 1:400 AB complex, Vectastain PK-6100 standard in TBS for 2 h at RT; (5) color development was initiated by the introduction of 100 µL 0.1% H2O2 to the 3,3′-diaminobenzidine  solution. The sections were rinsed multiple times with Tris buffered Saline (pH 7.4) between the above-described steps. The specificity of the primary antibody was tested with omitting the primary antibody in the protocol of the staining, which resulted in the absence of detectable immunostaining, and Western blot, which showed a band at 95 kDa. The optical density (OD) of the staining was measured with a Quantimet 550 image analysis system  as has been used before [Coronal brain sections (20 µm thick) were processed for immunohistochemical analysis of the post-synaptic marker PSD-95 with a free-floating technique according to the following steps: (1) incubation with 0.3% Hd before ,26. In tpost hoc test. The Kruskal-Wallis test was used for non-parametric data with the Mann-Whitney U test as a post hoc test. All statistical analyses were performed with the Statistical Package for Social Sciences (SPSS) .The distribution of all parameters was checked with the Shapiro-Wilk normality test. Normally distributed data were tested with a one-way ANOVA with the Bonferroni test as a df = 6; F = 64.181, post hoc Bonferroni analysis: High-Phe-Mid-Phe; p = 0.000 High-Phe-Low-Phe; p = 0.000, Mid-Phe-Low-Phe; p = 0.000, High-Phe with and without; p = 0.298, Mid-Phe with and without; p = 1.000, Low-Phe with and without; p = 1.000).Body weight and blood Phe concentrations were measured to monitor the dietary treatment. The absolute Phe intake, corrected for body weight, food intake, and the measured Phe in the food, confirmed that Phe intake was significantly different between the different Phe content groups but not between the different Phe content groups and their corresponding SNC groups ; p = 0.008). Post hoc testing revealed that PSD-95 OD in the DG-IB and DG-OB was significantly decreased in PKU mice fed with diets without SNC compared to WT by 54% and 64%, respectively . Although these significant differences were still present in DG-IB and DG-OB between the WT and PKU mice fed with diets with SNC, the decrease was considerably less: 29% and 27%, respectively . A clear trend was observed in the cornus ammonis 1 (CA1) and cornus ammonis 3 (CA3) cell layers , where PKU mice on diets without SNC showed a reduction in PSD-95 OD of 44% in the CA1 and 51% in the CA3 compared to WT mice. In the CA1, this difference was attenuated for the PKU mice fed with diets with SNC, as the difference between this group and WT mice was only 8%. In the striatum and prefrontal cortex, no significant differences were found between the groups ; no PKU PSD-95 expression deficit was present in PKU mice, and no change was induced by SNC.Non-parametric tests were used to examine the PSD-95 immunoreactivity differences. No significant differences were found between the Phe-content groups of PKU mice for all brain regions studied. Therefore, all PKU mice from the different Phe groups were pooled into two groups: those with and without SNC supplementation . The immunoreactivity of these three groups are depicted in To the best of our knowledge, this is the first report of reduced hippocampal PSD-95 expression in the PKU mouse model. The most important finding of our study is that SNC supplementation seems to dampen PKU PSD-95 OD deficits towards WT values in specific areas of the hippocampus. Therefore, this study indicates that SNC supplementation could have a positive effect on synaptic functioning by lessening the reduced expression of PSD-95 in the hippocampus of C57BL/6 PKU mice in the regions affected in PKU. This positive effect was most clear in the dendrites of the DG granular cells at the level of the cell layer, and to a lesser extent in CA1 pyramidal cell dendrites at the level of the cell layer.PSD-95 is a scaffolding protein which is post-synaptically present in glutamatergic and serotonergic synapses ,28. In get al. [et al. [The relation between PSD-95 and the glutamatergic AMPA and NMDA receptors suggests that the PKU-specific reduction in PSD-95 could specifically affect these receptors. However, the literature concerning this topic is somewhat contradictory. Martynyuk et al.  found a et al. . Despite [et al.  also repIn consensus with the literature, this study shows reduced levels of a protein associated with synaptic functioning in PKU mice ,13,14. ITo conclude, this study shows that SNC supplementation could have a positive effect on PSD-95 expression in the specific hippocampal subregions affected in C57BL/6 PKU mice. The examination of additional pre-and post-synaptic markers and functional outcomes, e.g., executive functioning, will be key to the subsequent more extensive investigation of synaptic dysfunction in PKU mice and the beneficial effects of SNC supplementation.